G−1:C73 Recognition by an Arginine Cluster in the Active Site of Escherichia coli Histidyl-tRNA Synthetase

Abstract

Aminoacylation of a transfer RNA (tRNA) by its cognate aminoacyl-tRNA synthetase relies upon the recognition of specific nucleotides as well as conformational features within the tRNA by the synthetase. In Escherichia coli, the aminoacylation of tRNA(His) by histidyl-tRNA synthetase (HisRS) is highly dependent upon the recognition of the unique G-1:C73 base pair and the 5'-monophosphate. This work investigates the RNA-protein interactions between the HisRS active site and these critical recognition elements. A homology model of the tRNA(His)-HisRS complex was generated and used to design site-specific mutants of possible G-1:C73 contacts. Aminoacylation assays were performed with these HisRS mutants and N-1:C73 tRNA(His) and microhelix(His) variants. Complete suppression of the negative effect of 5'-phosphate deletion by R123A HisRS, as well as the increased discrimination of Q118E HisRS against a 5'-triphosphate, suggests a possible interaction between the 5'-phosphate and active-site residues Arg123 and Gln118 in which these residues create a sterically and electrostatically favorable pocket for the binding of the negatively charged phosphate group. Additionally, a network of interactions appears likely between G-1 and Arg116, Arg123, and Gln118 because mutation of these residues significantly reduced the sensitivity of HisRS to changes at G-1. Our studies also support an interaction previously proposed between Gln118 and C73. Defining the RNA-protein interactions critical for efficient aminoacylation by E. coli HisRS helps to further characterize the active site of this enzyme and improves our understanding of how the unique identity elements in the acceptor stem of tRNA(His) confer specificity.

The requirement for the highly conserved G−1 residue of Saccharomyces cerevisiae tRNAHis can be circumvented by overexpression of tRNAHis and its synthetase

“…The predominant mechanism requires the G À1 residue or its phosphate as a crucial identity element for the corresponding histidyl-tRNA synthetase (HisRS), based on experiments in E. coli and yeast (Himeno et al 1989;Francklyn and Schimmel 1990;Rudinger et al 1994;Nameki et al 1995;Fromant et al 2000;Connolly et al 2004;Rosen and MusierForsyth 2004;Gu et al 2005;Rosen et al 2006). Since the G À1 residue is unique to tRNA His , there would be strong evolutionary pressure to maintain this recognition element once it had evolved.…”

“…Indeed, one welldocumented role of the G À1 residue is for aminoacylation of tRNA His . The G À1 residue or its phosphate is a strong identity element in vitro for histidylation of E. coli tRNA His and yeast tRNA His by their corresponding synthetases (Himeno et al 1989;Francklyn and Schimmel 1990;Rudinger et al 1994;Nameki et al 1995;Fromant et al 2000;Connolly et al 2004;Rosen and Musier-Forsyth 2004;Rosen et al 2006), and in vivo in yeast, based on the concomitant loss of G À1 from tRNA His and accumulation of deacylated tRNA His that occurs as Thg1 is depleted (Gu et al 2005). Thus, the G À1 residue may have been retained during evolution primarily as a determinant for histidyl-tRNA His synthetases.…”

Evolutionary conservation of a functionally important backbone phosphate group critical for aminoacylation of histidine tRNAs

“…Previous studies indicate that substitutions of the G-1:C73 determinant in the histidine system generally impose little or no effect on K M in the kinetics of aminoacylation (Himeno et al 1989;Yan et al 1996), or on the apparent dissociation constant (K D ), as determined by a competitive filter binding analysis (Bovee et al 1999). While a crystallographic model of the HisRS:tRNA His complex is not yet available, phylogenetics (Ardell and Andersson 2006), mutagenesis (Hawko and Francklyn 2001;Connolly et al 2004), and comparative modeling with the AspRS:tRNA Asp complex all support the prediction that the motif 2 loop of HisRS provides critical side-chains for reading out the identity/recognition elements on the tRNA His acceptor stem. By analogy to seryltRNA synthetase (Cusack et al 1996), which belongs to the same subclass of synthetases as HisRS (i.e., class IIa) (Cusack et al 1991), these side-chains are likely to undergo a small but functionally significant conformational change between the histidinol-ATP bound state and the tRNA bound state.…”

“…By analogy to seryltRNA synthetase (Cusack et al 1996), which belongs to the same subclass of synthetases as HisRS (i.e., class IIa) (Cusack et al 1991), these side-chains are likely to undergo a small but functionally significant conformational change between the histidinol-ATP bound state and the tRNA bound state. On the basis of published mutational analyses (Hawko and Francklyn 2001;Connolly et al 2004), and the observed covariation between the motif 2 loop and the ÿ1:73 base pair (Ardell and Andersson 2006), the interactions most critical for specificity in the prokaryotic HisRS system appear to be between Arg123 and the 59 phosphate, and between Gln118 and G-1:C73 (Connolly et al 2004). The substitution of Gln118 by a tripeptide of hydrophobic residues in the yeast and other eukaryotic HisRS systems may indicate a diminished role for polar contacts to G-1:A73.…”

Γονιδιωματική Ανάλυση Και Λειτουργικός Χαρακτηρισμός Ασύνηθων Συστημάτων Αμινοακυλίωσης Του Μεταφορικού RNA Σε Παθογόνους Μικροοργανισμούς

“…Χαρακτηριστικά παραδείγματα, η μεγάλη μεταβλητή θηλιά του tRNA Ser (Asahara et al, 1994), το νουκλεοτίδιο N-1 του tRNA His (Connolly et al, 2004) και το ασύνηθες ζεύγος βάσεων G15-G48 του tRNA Cys στο Escherichia coli (Hou et al 1993).…”